Seed storage protein is expressed in seeds only during the maturing stage, and the expression of genes encoding this protein is analyzed as a suitable model for investigating the transcription regulatory mechanism of plant genes (Goldberg, R. B. et al., Science 266: 605–614, 1994). The expression of a gene that codes for a seed storage protein is known to be regulated by the cooperation of a plurality of cis factors in a promoter. The binding of a transcription factor to a specific cis regulatory factor is important in the initiation of transcription and the tissue- and time-specific expression. It can be explained that the expression of a seed storage protein is induced by several types of cis regulatory factors relating to the regulation of seed-specific expression when transcription factors that recognize specific cis regulatory factor bind and aggregate. Functional analyses of cis regulatory factors and transcription factors of crop storage protein genes have been conducted in order to elucidate the molecular mechanism of the expression of seed storage proteins (Thomas, T. L., Plant Cell 5: 1401–1410, 1993; Morton, R. L. et al., in Seed Development and Germination, pp. 103–138, Marcel Dekker, Inc., 1995).
However, despite considerable research, analyses using transformed plants failed to identify the cis regulatory factors essential for gene expression regulation in nearly all crops studied, and the gene expression regulatory mechanism has still not been clearly understood. In the case of monocotyledons in particular, the promoter analyses using stable transformed plants has been performed in only the seed storage protein, glutelin, of the rice plants. On the other hand, in the case of maize, wheat and barley, analyses have been conducted using particle guns or tobacco transformants (Muller, M. and Knudsen, S., Plant J. 6:343–355, 1993; Albani, D. et al., Plant Cell 9: 171–184, 1997; Marzabal, P. M. et al., Plant J. 16: 41–52, 1998).
It has been shown that the endosperm-specific expression of the seed storage protein gene of grains is controlled by the collaborative action of several types of cis regulatory factors. The Prolamin box (TGTAAAG), GCN4 motif (TGA(G/C)TCA), AACA motif (AACAAAA), and ACGT motif, which are conserved in the seed storage protein gene promoters of numerous grains, have been characterized as cis regulatory factors involved in endosperm-specific expression by loss-of-function and gain-of-function analyses (Morton, R. L. et al., In: Seed Development and Germination, pp. 103–138, Marcel Dekker Inc., 1995).
The GCN4 motif has been frequently found not only from seed storage protein gene, but also from promoters of genes involved in the metabolism (Muller, M. and Knudsen, S., Plant J. 6: 343–355, 1993). Recently, a polymer of the GCN4 motif of rice plant glutelin gene has been found to reproduce endosperm-specific expression in transformed rice plants, and remarkable decrease in promoter activity and changes in its expression pattern have been found due to the substitution or deletion of nucleotides in the GCN4 motif. These facts prove that the GCN4 motif plays an important role in endosperm-specific expression (Wu, C. Y. et al., Plant J. 14: 673–683, 1998). The GCN4 motif is coupled to a Prolamin box (TGTAAAG) via a plurality of bases in many cases, and is one of the constituents of the two-factor endosperm box found in the prolamin gene promoters of nearly all grains, including wheat glutenin, barley hordein, rye secalin, sorghum cafulin and adlay coixin. The AACA motif is involved in the expression of nearly all rice glutelin genes. Although the combination of two motifs (GCN4 motif and Prolamin box or GCN4 motif and AACA motif) is required for gene expression, in order to adequately function as an endosperm-specific promoter, an additional motif is essential (Takaiwa, F. et al., Plant Mol. Biol. 30: 1207–1221, 1996; Yoshihara, T. et al., FEBS Letts. 383: 213–218, 1996; Wu, C. Y. et al., Plant J. (in press)). Recently, it has been demonstrated that, in order to function as aminimum promoter capable of reproducing endosperm-specific expression in glutelin genes (GluB1) of rice plant, at least three constituents, the GCN4 motif, the AACA motif, and the ACGT motif, present in the −197 bp promoter region, are essential (Wu, C. Y. et al., Plant J. 14: 673–683, 1998; Wu, C. Y. et al., Plant J. 23: 415–421, 2000).
Opaque2 (O2) of maize is an endosperm-specific transcription factor of the bZIP type, and this O2 binds to the ACGT motif in the 22 kDa α-zein gene promoter of maize to activate transcription (Schmidt, R. J. et al., Plant Cell 4: 689–700, 1992). O2 has been reported to be involved in endosperm-specific transcription of b-32 ribosome deactivating protein gene by binding to the (Ga/tTGAPyPuTGPu) sequence (Lohmer, S. et al., EMBO J. 10: 617–624, 1991). O2 is thus considered to have a wide-ranging binding capability. Reportedly, the GCN4 motif is recognized by O2, and transcription is activated through the binding of O2 to the GCN4 motif (Wu, C. Y. et al., Plant J. 14: 673–683, 1998; Holdsworth, M. J. et al., Plant Mol. Biol. 29: 711–720, 1995). In seeds, during the maturing stage, in vivo footprint analysis showed that the nuclear protein binds to the GCN4 motif and Prolamin box present in wheat low molecular weight glutenin gene promoter (Vicente-Carbajos, J. et al., Plant J. 13: 629–640, 1998) and maize γ-zein gene promoter (Marzabal, P. M. et al., Plant J. 16: 41–52, 1998). In addition, the results of an in vitro DNaseI footprint analysis showed that the nuclear protein of maturing rice plant seeds as well as GST-O2 fused protein specifically recognize the GCN4 motif of the rice glutelin gene promoter (Wu, C. Y., et al., Plant J. 14: 673–683, 1998; Kim, S. Y. and Wu, R., Nucl. Acids Res. 18: 6845–6852, 1990). These findings indicate that an O2-like transcription factor is present in grain seeds, and that it controls the endosperm-specific expression of numerous seed storage protein genes mediated by the GCN4 motif.
Recently, cDNA clones of transcription factors that recognize the GCN4 motif have been isolated in wheat (Albani, D. et al., Plant Cell 9: 171–184, 1997) and barley (Vicente-Carbajos, J. et al., Plant J. 13: 629–640, 1998; Onate, L. et al., J. Biol. Chem. 274: 9175–9182, 1999), and have been named SPA, BLZ1 and BLZ2. These transcription factors have been determined to activate the transcription of seed storage protein genes mediated by the GCN4 motif in wheat low molecular weight glutenin and barley B1 hordein gene promoter. Interestingly, these transcription factors were expressed seed-specifically. Although cDNA that codes for a transcription factor having a high homology with the bZIP domain of O2 has previously been isolated from rice plants, it remains to be confirmed whether or not it activates transcription of seed storage protein gene mediated by the GCN4 motif (Izawa, T. et al., Plant Cell 6: 1277–1287, 1994; Nakase, M. et al., Plant Mol. Biol. 33: 513–522, 1997).